The mammalian small intestine is lined by a highly differentiated epithelium comprised of specialized absorptive and secretory cell lineages that display a wide-ranging, yet tightly regulated diversity along the cephalo-caudal axis, resulting in an exquisite efficiency in the absorption of dietary nutrients and water. These functions, however, are easily disrupted by infection, inflammatory diseases such as Crohn's disease, or resection. Thus, for us to eventually develop strategies to regenerate lost or deficient intestinal function when gastrointestinal processes go awry, it is essential to understand the molecular events that are responsible for the development and maintenance of a normally functioning intestine. Over the past 5 yrs, we discovered essential roles for the evolutionarily conserved Gata family of transcription factors in the regulation of intestinal gene expression and cell fate allocation. Gata4, a transcription factor that is expressed in the proximal small intestine but not in the distal ileum, determines jejuno-ileal identities by activating and repressing specific absorptive enterocyte genes within the same cell. Gata4 and Gata6 are co-expressed in crypt cells of the proximal small intestine, and Gata4 or Gata6 is required for specification of the Paneth cell fate. Specific Aims: In Aim I, we will test the hypothesis that Gata4 is a universal regulator of jejunal-ileal differences in absorptive enterocyte gene expression using profiling analysis on the jejunum of conditional Gata4 null mice. We will also define the signature promoter characteristics of primary absorptive enterocyte target genes that are activated or repressed by Gata4. In Aim II, we will test the hypothesis that a committed Paneth/goblet progenitor requires Gata factors to progress to the Paneth lineage by defining the changes in intestinal morphology and lineage commitment, positioning, and gene expression that result from conditional Gata4 and Gata6 deletion. This will allow us to place Gata regulation in the genetic hierarchy of cell fate determination in the intestine. In Aim III, we will test the hypothesis that Friend of Gata (Fog) cofactors mediate some or all of the intestinal Gata function by defining the alterations in absorptive enterocyte gene expression and cell fate determination in the jejunum of mice in which Fog1, Fog2, or Fog1 and Fog2, are conditionally deleted in the small intestine. In Aim IV, we will test the hypothesis that Gata4 activates or represses specific absorptive enterocyte genes by recruitment of specific coactivators or corepressors that mediate histone acetylation or deacetylation, respectively, using in vivo chromosomal immunoprecipitation assays and our conditional null models for Gata4 and Fog cofactors. The results generated from the studies proposed in this application will further our understanding of intestinal differentiation, and allow us to develop interventions that restore intestinal function impaired by intestinal disease or resection. PUBLIC HEALTH RELEVANCE: The mammalian small intestine is lined by a highly differentiated epithelium comprised of specialized absorptive and secretory cell lineages that display a wide-ranging, yet tightly regulated diversity along the cephalo-caudal axis, resulting in an exquisite efficiency in the absorption of dietary nutrients and water. These functions, however, are easily disrupted by infection, inflammatory diseases such as Crohn's disease, or resection. Thus, for us to eventually develop strategies to regenerate lost or deficient intestinal function when gastrointestinal processes go awry, it is essential to understand the molecular events that are responsible for the development and maintenance of a normally functioning intestine. Over the past 5 yrs, the laboratory of the PI made an important discovery regarding our understanding of control mechanisms that determine specific functions within this organ. Gata4, a member of an evolutionarily conserved family of regulatory proteins, is expressed at high levels throughout most of the small intestine, but at low levels in distal part of the small intestine. Using state-of-the-art technology in which Gata4 was inactivated in the intestine, the middle part of the intestine (jejunum) where Gata4 is normally expressed underwent a transformation in which it became like the distal part (ileum). This finding revealed that Gata4 is required to activate specific genes while at the same time repressing others within the same cell. How this occurs is unknown, but understanding the underlying mechanisms is likely to provide opportunities for therapeutic advantage. A second key finding by the PI was the Gata4 or Gata6 are required for specific types of cells of the small intestine to develop normally. Understanding this process will allow us to develop strategies for manipulating these process for therapeutic gain. In the proposed studies, we will utilize a series of novel in vivo models together with contemporary gene regulation analysis to define lineage specification. The results of these studies will provide fundamental insight into the processes that maintain the mechanisms by which Gata factors regulate processes of gene expression and cell lineage determine normal functioning intestine enabling the future development of interventions designed to regenerate or repair lost or deficient function due to intestinal disease or resection.